Project 3: Influenza Virus HA and NA Immunogen Design

Abstract Developing next-generation vaccines for rapidly evolving pathogens (e.g., influenza, HIV) requires implementing rational approaches to engineer immunogens that can direct immune responses to conserved epitopes; in doing so, broad, durable responses are likely to occur. A “universal” or broadly protective influenza vaccine should induce broad immunity within circulating influenzas (e.g., H1, H3) and provide heterosubtypic and pre-pandemic protection. A goal of Project 3 is to design immunogens that elicit such protective humoral responses by targeting key conserved viral sites on the influenza hemagglutinin (HA) and neuraminidase (NA). We build upon our initial immunogen design strategies primarily focused on directing B-cell responses to the HA receptor binding site (RBS) to address how T-cell help influences the elicited immune responses. Furthermore, we integrate our immunogen design strategies established for HA and extend them to NA in order to understand if the design principles for directing immune responses to HA are generalizable. Specifically, we use our structure-guided “resurfacing” approach to graft the NA catalytic site from circulating N1 and N2 NAs onto exotic, avian non- circulating NAs. These “acceptor” NAs will serve as molecular scaffolds to present the conserved N1 and N2 catalytic site but with a heterologous periphery; elicited responses will maintain conserved contacts but adapt to the heterologous periphery and thus broadening the response. We use these resurfaced NAs to additionally develop NA heterochimeric tetramers (NAtChs) that present four copies of the grafted catalytic site but on four antigenically distinct scaffolds in efforts to increase immune focusing to this epitope. Finally, we will develop a novel viral-antibody coevolution assay using directed evolution platforms to understand how influenza may evolve to escape a focused immune response elicited by our engineered immunogens. This innovative approach may help anticipate viral escape and allow identification of vaccine regimen(s) that force influenza into an “evolutionary trap” by compromising overall viral fitness. Collectively, the data generated within this Project will contribute to developing next-generation influenza vaccines incorporating both HA and NA as potential immunogens.